Bombesin-related peptides ([gastrin-releasing peptide [GRP], neuromedin B) interact with two distinct receptors (GRP-R, NMB-R) to mediate a number of effects in the gastrointestinal tract (GI), central nervous sytem (CNS) and on growth of normal and neoplastic tissues. Furthermore, two related receptors, a mammalian orphan receptor (BRS-3), having 60% homology to GRP-R and a novel receptor in amphibians, BB-4-R has been described recently. The aims of this project are to understand the pharmacology, molecular pharmacology, and cell biology of these receptors as well as to develop specific agonists and antagonists that can be used to determine their physiological roles. Investigations being performed include expression of these receptors in stable cell lines that resemble native receptors in their cell biology and pharmacology; investigations using site-directed mutagenesis and receptor chimeras to define receptor structural determinants of ligand selectivity and specificity for agonists and antagonists, pharmacological studies of BN-related peptides to identify selective agonists/antagonists and studies of native cells and transfected cells to define the transduction cascades of these receptors. Recently we have reported that the peptoid PD168368 is the first high affinity NMBR antagonist with sufficient selectivity to be generally useful, that functions as an antagonist in all species including human. To investigate the molecular basis for PD 168368 selectivity for the NMBR, we made chimeric NMB-R and GRP-R as well as used site-directed mutagenesis. Our results show that in contrast to peptide antagonists, PD168368's selectivity is determined by amino acids in the transmembrane (TM)region of the receptor. Both loss of affinity and gain of affinity chimeric receptors support this conclusion and show it is localized in the 5th TM. Using site-directed mutagenesis, the Tyr220 in the NMBR compared to Phe in GRPR was found to account for selectivity by PD168368. Computer modeling of the ligand and receptor suggest the hydroxyl of Tyr220 interacts by hydrogen bonding with the nitrophenyl group of PD168368 and a binding model has been proposed.